: Antibodies directed against allograft antigens are being increasingly recognized as critical determinants of allograft outcomes in adults. Pediatric heart transplant candidates are frequently allosensitized based on prior exposure to blood products and homografts. To overcome the high wait-list mortality, transplantation across a donor-specific cross-match has recently been performed in several pediatric centers, with early encouraging results. This application brings together a group of six leading heart transplant centers and leading transplantation scientists to study the impact of preformed and de novo alloantibodies on pediatric heart transplant outcomes. Specific Aim 1 will define the prevalence, titer and antigen specificities of anti- HLA and MICA alloantibodies, in a consecutive cohort of 380 pediatric transplant candidates. Risk factors for their development will be determined. Candidates with preformed anti-HLA antibodies and a positive donor- specific cross-match will enter into a multi-center, non-randomized treatment trial to evaluate the safety and efficacy of a uniform protocol of management (Specific Aim 2). The protocol will involve intra- and post- operative plasma exchange/pheresis and a uniform immunosuppressive protocol. The primary end-point will be the proportion of patients who are free of death, graft loss and rejection with hemodynamic compromise at one year after transplantation. We hypothesize that 75% of patients will achieve this end-point. Non- sensitized candidates will be studied within a prospective observational protocol under a uniform immunosuppressive regimen (Specific Aim 3) to determine the prevalence of de novo anti-HLA and anti- MICA antibodies. We hypothesize that the development of de novo antibodies will be an independent predictor of more frequent and severe acute rejections. In Specific Aim 4, mechanistic studies will be performed to seek explanation for why some children develop graft injury while others appear to 'accommodate' their graft in the presence of a positive donor-specific cross-match. We hypothesize that three interrelated factors contribute to graft accommodation: 1). Characteristics of the antibody(s) 2). Endothelial cell resistance to antibody mediated damage and 3). Replacement of donor vascular endothelial cells with those of recipient origin leading to endothelial chimerism. Finally, we will investigate the development of a non-invasive marker of humoral rejection; quantitative measurement of cell bound complement activation products. The CTOT-C provides an ideal mechanism for performing the proposed studies and should lead to important new knowledge about antibody mediated graft injury in pediatric heart transplantation. It should provide much needed information for the design of future clinical trials in this field. Relevance: Pediatric heart transplantation remains palliative with high demand for re-transplantation and great economic and social burden. All strategies that could enhance allograft survival will improve the health status of these children and will relieve the burden on the limited donor pool.